VOL.
8,
NO.
5,
MAY
1969
Perturbation of Proton and Detergent Binding Sites in Bovine Serum Albumin by Acetimidation" Joseph Avruch,t Jacqueline A. Reynolds,$ and John H. Reynolds8
ABSTRACT: Modification of bovine serum albumin by blocking the 57 lysine residues with methyl acetimidate hydrochloride leads to a protein with identical circular dichroism spectra with that of the native macromolecule but a slightly increased intrinsic viscosity. The binding isotherm of sodium dodecyl sulfate to both protein
T
he lysine residues of bovine serum albumin have been implicated in stabilization of the native state through charge interaction with the 40 buried carboxylate ions (Vijai and Foster, 1967; Foster and Clark, 1962) and in the binding sites on bovine serum albumin for sodium dodecyl sulfate (Markus et at., 1964). The last-named authors showed that sodium dodecyl sulfate stabilized native bovine serum albumin against urea denaturation but did not stabilize a modified form of bovine serum albumin in which the lysines were blocked to give an uncharged side chain. Tyrosine and tryptophan also have been shown to be involved in detergent binding to bovine serum albumin by Ray et at. (1966), Polet and Steinhardt (1968), and Reynolds et al. (1967) who showed spectral shifts in these aromatic residues as the result of binding C&la sulfate and sulfonate half-esters and C8-C12carboxylic acids to bovine serum albumin. The object of the present work was to block all lysine residues of bovine serum albumin with methyl acetimidate hydrochloride (Habeeb, 1966; Wofsy and Singer, 1963) to form a modified protein with structure NH2+
I/
PwNHZ+ +PwNHCCH, In so doing, the positive charge is left on all modified lysine residues but a longer side chain has been inserted. If €-amino groups are involved in charge-charge interactions with carboxylate groups and are also part of a set of binding sites for detergent ligands, it is reasonable to expect an alteration in both detergent and proton
* From the Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri 63 110, and Research Center, Monsanto Company, St. Louis, Missouri 63141. ReceivedJanuary 6, 1969. t Present address: Department of Internal Medicine, Washington University Medical School. 3 Department of Microbiology, Washington University Medical School. $ Research Center, Monsanto Co.: to whom reprint requests should be directed.
species is identical. However, the binding-induced difference spectra are reduced in magnitude when the ligand interacts with the modified protein. Proton binding studies indicate a larger number of carboxylate and tyrosine groups are exposed in acetimidated bovine serum albumin than in native bovine serum albumin.
binding properties of the modified protein. Previous studies of a limited nature (Habeeb, 1966) showed a small increase in the frictional ratio (
